DESC:FIELD OF THE INVENTION
The present invention relates to an improved enzymatic synthesis of Sitalgiptin or its pharmaceutically acceptable salts in good yield and purity. The present invention provides novel intermediate (R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5).

BACKGROUND OF THE INVENTION
Sitagliptin, sold under the brand name Januvia among others, is an anti-diabetic medication used to treat type 2 diabetes. Sitagliptin, is chemically, (R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine, with the formula;

Several processes are described in the art to introduce the ß-amino acid structure into the molecule of Sitagliptin. WO03/004498 uses an unusual chiral dihydropyrazine promoter, diazomethane and silver salts for introduction of ß-
amino acid structure as shown in Scheme 1 below:

However, the use of the reagents disclosed is undesirable on industrial scale.

In one of the approaches, a heterocycle is coupled to the ß-amino acid in later steps. The corresponding ?-aryl-3-amino acids are readily available from the corresponding ß-keto acids, prepared from acetic acids and malonic derivatives (WO 09/064476, WO10/122578, WO10/131025, WO 1 1/127794). The amino group however needs to be protected before coupling with the heterocycle in order to eliminate side reactions. The protection/deprotection steps considerably prolong the synthesis of Sitagliptin. (Scheme2).

Since then, several methods are developed in the art to introduce ß-amino acid structure into the molecule of sitagliptin which would be scalable on industrial level. The modifications include enantioselective reduction (WO 09/064476, WO 10/078440), by introducing chiral protecting groups with further diastereoselective crystallization (CN102126976), by crystallization of diastereomeric salt of compounds with chiral acids (WO 10/122578, WO 10/131025, J. Chem. Res. (4), 230 (2010)), or by introduction of a chiral center via natural source, such as aspartic acid derivatives (WO 1 1/035725, WO 1 1/1 16686A2, CN 102093245, CN 10212697) or by enzymatic approach.
The enzymatic process represents a promising approach and the present invention provides an enzymatic route for preparation of Sitagliptin with improved yield and purity.

SUMMARY OF THE INVENTION
In accordance with the above, the present invention provides an enzymatic synthesis of Sitagliptin which comprises the steps of;
i. Treating Racemate methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoic acid or its ester (1) in solvent with D or (R)-O-Acetyl mandelic acid (9) and 1-50% w/w CAL B enzyme, in presence of the catalyst under hydrogen pressure to obtain (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2);
ii. Hydrolyzing the compound of formula (2) in solvent with the base to obtain (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5);
iii. Reacting the compound (5) with 3-(Trifluoromethyl)-1,2,4-triazolo-[4,3-a] piperazine hydrochloride (7) in presence of coupling agent, solvent and the base to yield N-Acetyl Sitagliptin (10); and
iv. Converting N-Acetyl Sitagliptin (10) to its hydrochloride or phosphate salt.

In an aspect, the intermediate compound, (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) is hydrolyzed with acid to give methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (3).

In an aspect, the present invention provides the intermediate, (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5) of the formula;

DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described in detail with its preferred and optional embodiments so that the various aspects therein can be more clearly understood and appreciated.

In an embodiment, the present invention relates to an improved and efficient enzymatic synthesis of Sitagliptin which comprises the steps of;
i. Treating Racemate methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoic acid or its ester (1) in solvent with D or (R)-O-Acetyl mandelic acid (9) and 1-50% w/w CAL B enzyme, in presence of the catalyst under hydrogen pressure to obtain (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2);
ii. Hydrolyzing the compound of formula (2) in solvent with the base to obtain (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5);
iii. Reacting the compound (5) with 3-(Trifluoromethyl)-1,2,4-triazolo-[4,3-a] piperazine hydrochloride (7) in presence of coupling agent, solvent and the base to yield N-Acetyl Sitagliptin (10); and
iv. Converting N-Acetyl Sitagliptin (10) to its hydrochloride or phosphate salt.

In another embodiment, the intermediate compound, (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) is hydrolyzed with acid to give methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (3).

In an embodiment of the process step (i), the catalyst is selected from 1-20%w/w Raney nickel or 5-10% Palladium on Carbon. The hydrogen pressure is in the range of 1-15 kgs. The solvent is selected from aromatic hydrocarbon(s) such as toluene or xylene; lower alcohols such as methanol or ethanol; halogenated hydrocarbon, THF alone or mixtures thereof.

In another embodiment, the base for the hydrolysis is step (ii) is selected from alkali metal hydroxide or its hydrate, preferably the base is Lithium hydroxide monohydrate. The solvent for the reaction is selected from polar aprotic solvent such as THF, dioxane, diethyl ether and the like alone or mixtures thereof.

In another embodiment, the coupling agent for process step (iii) is selected from 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide(DIC) and the like. The base is selected from ethylamine, triethylamine, pyridine and the like. The solvent is selected from halogenated hydrocarbons or polar aprotic solvent comprising THF, dioxane, diethyl ether and the like alone or mixtures thereof.

In an embodiment of the process step (iv), N-acetyl Sitagliptin is converted to its hydrochloride salt using conc. hydrochloric acid or to its phosphate salt using phosphoric acid in presence of polar organic solvent such as lower alcohol and water alone or mixtures thereof.

In another embodiment, the acid for conversion of (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) to methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (3) is carried out using mineral acid.

The process of the present invention is represented in Scheme 1 below:
In an aspect, the present invention provides the intermediate, (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5) of the formula;

In an embodiment, the biocatalyst CAL B TA 1000-10000 u/gm and the CAL B enzyme 10000u/gm used in the process of the present invention expressed in Pichia pastoris is prepared in house. The enzymes used for biocatalyst are immobilized on polyacrylate supports. FERMENTA BIOTECH LTD manufactured IMMOBILIZED Biocatalyst CALB TA 1000- 10000 u/g, Covalent resins, expressed in Pichia pastoris. The LIQUID CAL B gene was heterologous expressed in Pichia pastoris. Large quantities of the CAL B enzyme were produced by fermentation. The fermentation broth containing the enzyme was concentrated to an activity of 15000-20000 TBU/ml manufactured by Fermenta Biotech Ltd. The Liquid biocatalyst CALB 10L (NLT 10000 TBU/mL is also manufactured by Fermenta Biotech Ltd.

In an embodiment of the present process, the enzyme (biocatalyst) is recycled and reused.

Examples:
Example 1: Step (i): General method of preparation of Methyl -(R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2)

1 equivalent of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 3-10 volumes of Toluene or Tetrahydrofuran with 1-1.5 equivalents of D or (R)-O-Acetyl mandelic acid (9) and 1-50% w/w immobilized CAL B enzyme,1-20% w/w Catalyst Raney Nickel or 1-10% w/w of 5-10% Palladium on Carbon Catalyst under Hydrogen pressure of 1-15 kgs at 60-80°C for 17-50 hours. After 20 hours TLC analysis indicated completion of reaction. The reaction mass was filtered, washed with toluene. The toluene layer was washed with water, 10% Aqueous Sodium bicarbonate solution and finally with water and purified by column chromatography. The 1H NMR, C13 NMR and MASS SPECTRA indicated the formation of the desired product i.e. Methyl -(R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2).

Example 1A:
10gms (0.0404moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Toluene with 9.6 gms of D or (R)-O-Acetyl mandelic acid (9) and 1gm (10% w/w of substrate 1)Immobilized CAL B enzyme, 2 gms of 10% Palladium on Carbon Catalyst under Hydrogen pressure of 10 kgs at 60-80°C for 17-24 hours. After 20 hours TLC analysis indicated completion of reaction. The reaction mass was filtered, washed with toluene. The Toluene layer was washed with 100 ml (4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water, solvent evaporated under vacuum and the residue was purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 4.6 gms
% Yield: 34.2%
The 1HNMR, C13NMR and mass spectra indicated the formation of the desired product i.e. Methyl -(R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2).

Example 1B:
10 gms (0.0429moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Toluene with 9.6 gms of D or (R)-O-Acetyl mandelic acid (9) and 1gm (10% w/w of substrate 1) Immobillized CAL B enzyme, 6 gms of Raney Nickel Catalyst under Hydrogen pressure of 10 kgs at 60-80°C for 17-24 hours. After 20 hours TLC analysis indicated completion of the reaction. The reaction mass was filtered, washed with Toluene, The Toluene layer was washed with 100 ml (4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water. The solvent evaporated under vacuum and the residue was purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 6.8 gms
% Yield: 58%
The 1H NMR, C13NMR and mass spectra indicated the formation of the desired product i.e. Methyl -(R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2).

Example 1C:
10 gms (0.0429moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Toluene with 9.6 gms of D or (R)-O-Acetyl mandelic acid (9) and 4gm (40% w/w of substrate 1) Immobilized CAL B enzyme, 6 gms of Raney Nickel Catalyst under Hydrogen pressure of 10 kgs at 60-80°C for 35-45 hours. After 44 hours TLC analysis indicated completion of reaction. The reaction mass was filtered, washed with Toluene. The Toluene layer was washed with 100 ml(4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water. The solvent evaporated under vacuum and the residue and purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 8 gms
% Yield: 68 %
The 1H NMR, C13NMR and mass spectra indicated the formation of the desired product i.e. Methyl -(R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2).

Example 1D:
10 gms (0.0429moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Tetrahydrofuran, 9.6gms of D or (R)-O-Acetyl mandelic acid (9) and 1 gm (10% w/w of substrate 1) Immobilized CAL B enzyme, 6 gms of Raney Nickel Catalyst under Hydrogen pressure of 5 kgs at 57-62°C for 17-24 hours. After 20 hours TLC analysis indicated completion of the reaction. The reaction mass was filtered, washed with Tetrahydrofuran. The Tetrahydrofuran was evaporated under vacuum and the residue was dissolved in 500 ml Ethyl acetate. The ethyl acetate layer was washed with 100 ml (4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water. The solvent evaporated under vacuum and the residue was purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 7.9 gms
% Yield: 68%

Example 1E
10 gms (0.0429moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Tetrahydrofuran, 9.6 gms of D or (R)-O-Acetyl mandelic acid (9) and 0.75 gm (7.5% w/w of substrate 1) Immobilized CAL B enzyme, 6 gms of Raney Nickel Catalyst under Hydrogen pressure of 5 kgs at 57-62°C for 17-40 hours. After 36 hours, TLC analysis indicated completion of the reaction. The reaction mass was filtered, washed with Tetrahydrofuran, The Tetrahydrofuran was evaporated under vacuum and the residue was dissolved in 500 ml Ethyl acetate. The ethyl acetate layer was washed with 100 ml (4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water. The solvent evaporated under vacuum and the residue was purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 7.1 gms
% Yield: 61%

Recycled Enzyme:

The Immobilized Enzyme CAL B from all the above examples were recovered and reused in the subsequent experiments. 10% w/w fresh enzyme CAL B(10%w/w of the total enzyme to be added in the experiment) was also added.
The examples are given below:

Example 1F
10 gms (0.0429moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Tetrahydrofuran, 9.6 gms of D or (R)-O-Acetyl mandelic acid (9) and 1.1 gm (11% w/w of substrate 1, 1gm recovered enzyme+0.1gm fresh enzyme) Immobilized CAL B enzyme, 6 gms of Raney Nickel Catalyst under Hydrogen pressure of 5 kgs at 57-62°C for 17-34 hours. After 30 hours TLC analysis indicated completion of the reaction. The reaction mass was filtered, washed with Tetrahydrofuran, The Tetrahydrofuran was evaporated under vacuum and the residue was dissolved in 500 ml Ethyl acetate. The ethyl acetate layer was washed with 100 ml (4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water. The solvent evaporated under vacuum and the residue was purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 7.6 gms
% Yield: 65%

Example 1F:
10gms (0.0404moles) of Racemate Methyl-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (1) was treated in 100 ml Toluene with 9.6 gms of D or (R)-O-Acetyl mandelic acid (9) and 1.1gm (11% w/w of substrate 1, 10% fresh enzyme +0.1gms of recovered Enzyme) Immobilized CAL B enzyme, 2 gms of 10% Palladium on Carbon Catalyst under Hydrogen pressure of 10 kgs at 60-80°C for 17-45 hours. After 40 hours TLC analysis indicated completion of reaction. The reaction mass was filtered, washed with Toluene, The Tolune layer was washed with 100 ml (4*25 ml) water, 50 ml (5*10 ml) 10% Aqueous Sodium bicarbonate solution and finally with 100 ml (4*25ml) water, solvent evaporated under vacuum and the residue was purified by column chromatography using silica gel of 60-200 mesh and Ethyl acetate: Methanol 95: 5 to 90: 10 ratios respectively as eluents to isolate the pure product.
Yield: 4.4 gms
% Yield: 32%

Example 2: Step (ii) General method for Preparation of (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5).

1 equivalent of Methyl -(R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) was dissolved in 2-10 volumes of Tetrahydrofuran. 2-4 equivalents of Lithium Hydroxide monohydrate in 1-3 volumes of water was added at 25-30°C. The reaction mass was stirred till the TLC analysis indicated the completion of reaction. The solvent, THF was evaporated under vacuum, 4-10 volumes of Ethyl acetate was added, and the aqueous layer was separated. The Organic layer was evaporated under vacuum and the crude compound was purified by column chromatography to isolate (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5).

Example 2A:
1gm (0.00346moles) of Methyl-(R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) was dissolved in 20 ml Tetrahydrofuran. 610 mg (0.0145moles) of Lithium Hydroxide monohydrate in 3ml of water was added at 25-30°C to the mixture. The reaction mass was stirred till the TLC analysis indicated the completion of reaction. The solvent, THF was evaporated under vacuum, 10ml Ethyl acetate was added, and the aqueous layer was separated. The Organic layer was evaporated under vacuum and the crude compound was purified by column chromatography using silica gel 60-200 mesh and Ethyl acetate: methanol 96: 4 to 90:10 ratios respectively as eluent to isolate (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5).
Yield: 0.86gm
% Yield: 90%

Example 2B:
10gm (0.0346moles) of Methyl-(R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) was dissolved in 200 ml Methanol. 6.1 mg (0.145moles) of Lithium Hydroxide monohydrate in 30 ml of water was added at 25-30°C to the mixture. The reaction mass was stirred till the TLC analysis indicated the completion of reaction. The solvent, methanol was evaporated under vacuum, 100 ml Ethyl acetate was added, and the aqueous layer was separated. The Organic layer was evaporated under vacuum and the crude compound was purified by column chromatography using silica gel 60-200 mesh and Ethyl acetate: methanol 96: 4 to 90:10 ratios respectively as eluent to isolate (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5).
Yield: 9 gms
% Yield: 94%

Example 3: Step (iii) General method for preparation of N-Acetyl Sitagliptin (10).
1 equivalent of (R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5) was dissolved in 2-100 volumes of Dichloromethane or Tetrahydrofuran. 2-2.5 equivalents of 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride, 3 equivalents of Triethylamine and 1.2 equivalents of 3-(Trifluoromethyl)-1,2,4-triazolo-[4,3-a]piperazine hydrochloride (7) were added and the reaction mass was stirred for 2-40 hours or till the TLC analysis indicated the completion of reaction. The solvent was evaporated, and the residue was dissolved in Ethyl acetate, washed with 10 volumes of water. After evaporation of the solvent the residue was purified by column chromatography to isolate N-Acetyl Sitagliptin (10). 1H NMR, C13NMR and Mass analysis indicated the formation of the desired compound.

Example 3A:
1 gm (0.00364moles) of (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5) was dissolved in 40 ml Tetrahydrofuran. 1.73 gms (0.0091 moles) of 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride, 1.11 gms (0.01092 moles) of Triethylamine and 0.9980 gms(0.00438 moles) of 3-(Trifluoromethyl)-1,2,4-triazolo-[4,3-a]piperazine hydrochloride (7) were added and the reaction mass was stirred for 25 hours or till the TLC analysis indicated the completion of reaction. The solvent was evaporated, and the residue was dissolved in Ethyl acetate, washed with 10 volumes of water. After evaporation of the solvent the residue was purified by column chromatography using silica gel 60-200 mesh and Ethyl acetate : Methanol 95: 5 to 85: 15 ratios respectively as eluent to isolate N-Acetyl Sitagliptin (10).
Yield: 0.85 gms
% Yield: 52%

Example 3B:
10 gm (0.0364moles) of (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5) was dissolved in 500 ml Dichloromethane. 17.3 gms (0.091 moles) of 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride, 11.1 gms (0.1092 moles) of Triethylamine and 9.98980 gms(0.0438 moles) of 3-(Trifluoromethyl)-1,2,4-triazolo-[4,3-a]piperazine hydrochloride (7) were added and the reaction mass was stirred for 25 hours or till the TLC analysis indicated the completion of reaction. The solvent was evaporated, and the residue was dissolved in Ethyl acetate, washed with 10 volumes of water. After evaporation of the solvent the residue was purified by column chromatography using silica gel 60-200 mesh and Ethyl acetate : Methanol 95: 5 to 85: 15 ratios respectively as eluent to isolate N-Acetyl Sitagliptin (10).
Yield: 12.3 gms
% Yield: 75.4%

Example 4: Step (iv) General method for preparation of Sitagliptin Phosphate or Hydrochloride.
1 equivalent of N-Acetyl sitagliptin was treated with either 2-10 volumes of concentrated Hydrochloric acid or 2-10 volumes of Phosphoric acid at 40-70°C till TLC analysis indicated completion of reaction. The excess acid was evaporated under vacuum and the residue was purified in water or isopropanol to isolate either Sitagliptin Hydrochloride or Phosphate.

Example 4A:
1gm (0.222 millimoles) of N-Acetyl sitagliptin was treated with 10 ml concentrated Hydrochloric acid at 70°C till TLC analysis indicated completion of reaction. The excess acid was evaporated under vacuum and the residue was purified in 45 ml Isopropanol to get the desired compound Sitagliptin Hydrochloride.
Yield: 0.78 gms
% Yield: 80%
1H NMR, C13NMR and Mass analysis indicates the formation of the desired compound.

Example 4B:
1gm (0.222 millimoles) of N-Acetyl sitagliptin was dissolved in 15ml of Isopropanol and 15 ml water, treated with 5 ml of Phosphoric acid drop wise in 20 minutes. The reaction mass was heated at 70°C till TLC analysis indicated completion of reaction. The slurry was filtered and washed with 15ml of Isopropanol, dried under vacuum to isolate pure Sitagliptin Phosphate.
Yield: 0.9 gms
% Yield: 80%
1H NMR, C13NMR and Mass analysis indicates the formation of the desired compound.

Example 5: General method of preparation of Methyl- (R)-3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (3).
1 equivalent of Methyl-(R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) was treated with 3-10 volumes of concentrated Hydrochloric acid, heated at 45-100°C for 2-10 hours till the TLC analysis indicated the completion of the reaction. After work up the crude residue was purified by column chromatography to isolate the desired compound- (R)-3-Amino-4-(2,4,5-Trifluorophenyl) butanoic acid. The 1H NMR, C13 NMR and mass spectra indicated the formation of the desired product i.e. Methyl- (R)- 3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (3).

Example 5A:
1gm (0.346 millimole) of Methyl-(R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) was treated with 10ml of concentrated Hydrochloric acid, heated at 45-100°C for 2-10 hours till the TLC analysis indicated the completion of the reaction. After work up the crude residue was purified by column chromatography using silica gel 60-200 mesh and Ethyl acetate: methanol 98:2 to 90: 10 ratios respectively as eluent to isolate the desired compound- (R)-3-Amino-4-(2,4,5-Trifluorophenyl) butanoic acid(3).
Yield: 0.7 gms
% Yield: 81 %.

Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.
,CLAIMS:
1. An improved enzymatic process for synthesis of Sitagliptin or its pharmaceutically acceptable salts comprising;
i. Treating Racemate methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoic acid or its ester (1) in solvent with D or (R)-O-Acetyl mandelic acid (9) and 1-50%w/w CAL B enzyme, in presence of the catalyst under hydrogen pressure to obtain (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2);
ii. Hydrolyzing the compound of formula (2) in solvent with the base to obtain (R)-3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5);
iii. Reacting the compound (5) with 3-(Trifluoromethyl)-1,2,4-triazolo-[4,3-a] piperazine hydrochloride (7) in presence of coupling agent, solvent and the base to yield N-Acetyl Sitagliptin (10); and
iv. Converting N-Acetyl Sitagliptin (10) to its hydrochloride or phosphate salt.

2. The enzymatic process as claimed in claim 1, wherein the Immobilized CAL B enzyme of step (i) is the biocatalyst CAL B TA 1000-10000 u/gm and the CAL B enzyme 10000u/gm expressed in Pichia pastoris and immobilized on polyacrylate supports.

3. The enzymatic process as claimed in claims 1 to 3, wherein the enzyme is recovered and reused.

4. The enzymatic process as claimed in claim 1, wherein (a) the catalyst of step (i) is selected from 1-20%w/w Raney nickel or 5-10% Palladium on Carbon; and (b) the solvent is selected from toluene, xylene, ethanol, methanol, tetrahydrofuran, dichloromethane or isopropanol.

5. The enzymatic process as claimed in claim 1, wherein (a) the base for the hydrolysis in step (ii) is selected from alkali metal hydroxide or its hydrate; and (b) the solvent is selected from THF, dioxane, diethyl ether and the like alone or mixtures thereof.

6. The enzymatic process as claimed in claim 1, wherein (a) the coupling agent for step (iii) is selected from 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), N,N'-dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide(DIC); (b) the base is selected from ethylamine, N,N-Diisopropylethyl amine, triethylamine or pyridine; (c) the solvent is selected from halogenated hydrocarbons or polar aprotic solvent comprising THF, dioxane, diethyl ether alone or mixtures thereof.

7. The enzymatic process as claimed in claim 1, wherein the acid for step (iv) is selected from hydrochloric acid or phosphoric acid.

8. The enzymatic process as claimed in claim 1, wherein (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoate (2) is converted to methyl 3-Amino-4-(2,4,5-Trifluorophenyl) butanoate (3) using mineral acid.

9. The intermediate (R)- 3-Acetamido-4-(2,4,5-Trifluorophenyl) butanoic acid (5) comprising;